DETEKSI MUTASI INTRON 1 IVS-1 NT 5 (G>C) GEN PENGKODE BETA GLOBIN PADA CARRIER BETA THALASSEMIA DENGAN PCR-RFLP

LILY WIDYAWATI, Dr. Niken Satuti N Handayani, M.Sc.;Dr.biol.hom. Nastiti Wijayanti, M.Si.;Ganies Riza Aristya, S.Si., M.Sc.

2017 | Skripsi | S1 BIOLOGI

Penelitian ini dilakukan untuk mengetahui keberadaan mutasi IVS I nt 5 pada sejumlah sampel darah yang secara hematologis didiagnosis sebagai carrier beta thalassemia. Carrier beta thalassemia adalah suatu penyakit herediter yang tidak dapat diamati dengan kenampakan luar karena pada kondisi heterozigot bersifat asimtomatik. Dalam pendataan sampel darah carrier beta thalassemia, peneliti bekerja sama dengan POPTI dan Laboratorium Klinik Prodia untuk melihat data hematologis setiap sampel darah seperti hematologi rutin (Cell Blood Count, CBC), nilai indeks korpuskular (Mean Corpuscular, MC), analisis hemoglobin dengan metode High Performance Liquid Chromatography (HPLC), dan gambaran darah tepi. Pada penelitian ini digunakan sampel berupa frozen blood 9 individu yang berdasarkan analisis hematologis terdiagnosis sebagai carrier beta thalassemia. DNA sampel darah diisolasi untuk selanjutkan dilakukan amplifikasi DNA sekuen yang spesifik menggunakan set primer spesifik sehingga menghasilkan amplikon berukuran 293 bp (basepairs). Selanjutnya DNA hasil amplifikasi tersebut diinkubasi dengan enzim restriksi Cac8I pada suhu 370C selama 6 jam di dalam waterbath. Interpretasi data dilakukan dengan membandingkan perbedaan ukuran dan jumlah fragmen digesti pada sampel normal (kontrol negatif) dengan sampel carrier beta thalassemia serta membandingkan persamaan ukuran dan jumlah fragmen antara sampel kontrol positif (mutasi IVS I nt 5) dengan sampel carrier beta thalassemia. Hasil penelitian menunjukkan 6 dari 9 individu carrier beta thalassemia dideteksi mengalami mutasi IVS I nt 5 dengan PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Sehingga dapat disimpulkan bahwa dengan PCR-RFLP dapat dideteksi terdapat 6 dari 9 individu carrier beta thalassemia mengalami mutasi pada IVS I nt 5 (G>C).

This study was conducted to determine the presence of IVS mutations I nt 5 in a number of blood samples haematologically diagnosed as a carrier beta thalassemia. Carrier beta thalassemia is a hereditary disease whose characteristics can not be observed with outside appearance because the condition of heterozygotes is asymptomatic. In data collection of blood samples diagnosed with beta thalassemia carrier, the researchers collaborated with POPTI and Prodia Clinical Laboratory to view hematological data of each blood sample such as routine hematologic (Cell Blood Count, CBC), corpuscular index value (Mean Corpuscular, MC), hemoglobin analysis with methods of High Performance Liquid Chromatography (HPLC), and peripheral blood images. This study used a sample of frozen blood 9 individuals based on hematologic analysis which diagnosed as a carrier beta thalassemia. The DNA of blood sample was isolated for further specific DNA sequence amplification using a specific primer set to produce a 293 bp amps (basepairs). Then the amplified DNA was incubated with restriction enzyme Cac8I at 37 0C for 6 hours in the waterbath. Interpretation of data was done by comparing the difference in size and number of digestive fragments in normal / negative control samples with samples diagnosed with beta thalassemia carrier and comparing the equation of size and number of fragments between positive control samples of IVS mutation I nt 5 with the samples diagnosed with beta thalassemia carrier. The results showed 6 of 9 individuals diagnosed with beta thalassemia carrier were detected by IVS I nt 5 mutation by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technique. So it can be concluded that 6 of 9 individual carrier beta thalassemia have mutation in intron one that is IVS-I nt 5.

Kata Kunci : Kata kunci : β-thalassemia, RFLP, mutasi, enzim restriksi, hematologis; Keywords: β-thalassemia, RFLP, mutation, restriction enzyme, hematologic